Time-dependent changes in expression of troponin subunit isoforms in unloaded rat soleus muscle

This study focuses on the effects ofmechanical unloading of rat soleus muscle on the isoform patterns ofthe three troponin (Tn) subunits: troponin T (TnT), troponin I (TnI),and troponin C (TnC). Mechanical unloading was achieved by hindlimbunloading (HU) for time periods of 7, 15, and 28 days. Relativeconcentrations of slow and fast TnT, TnI, and TnC isoforms wereassessed by electrophoretic and immunoblot analyses. HU inducedprofound slow-to-fast isoform transitions of all Tn subunits, althoughto different extents and with different time courses. The effectivenessof the isoform transitions was higher for TnT than for TnI and TnC.Indeed, TnI and TnC encompassed minor partial exchanges of slowisoforms with their fast counterparts, whereas the expression patternof fast TnT isoforms (TnTf) was largely increased after HU. Moreover, slow and fast isoforms of the different Tn were not affected in thesame manner by HU. This suggests that the slow and fast counterparts ofthe Tn subunit isoforms are regulated independently in response to HU.The changes in TnTf composition occurred in parallel with previouslydemonstrated transitions within the pattern of the fast myosin heavychains in the same muscles.

     

Distribution of myosin heavy chain isoforms in non-weight-bearing rat soleus muscle fibers

Talmadge, Robert J., Roland R. Roy, and V. Reggie Edgerton.Distribution of myosin heavy chain isoforms in non-weight-bearing rat soleus muscle fibers. J. Appl.Physiol. 81(6): 2540-2546, 1996.The effects of14 days of spaceflight (SF) or hindlimb suspension (HS) (Cosmos 2044)on myosin heavy chain (MHC) isoform content of the rat soleus muscleand single muscle fibers were determined. On the basis ofelectrophoretic analyses, there was a de novo synthesis of type IIx MHCbut no change in either type I or IIa MHC isoform proportions aftereither SF or HS compared with controls. The percentage of fiberscontaining only type I MHC decreased by 26 and 23%, and the percentageof fibers with multiple MHCs increased from 6% in controls to 32% inHS and 34% in SF rats. Type IIx MHC was always found in combinationwith another MHC or combination of MHCs; i.e., no fibers contained typeIIx MHC exclusively. These data suggest that the expression of thenormal complement of MHC isoforms in the adult rat soleus muscle isdependent, in part, on normal weight bearing and that the absence ofweight bearing induces a shift toward type IIx MHC protein expression in the preexisting type I and IIa fibers of the soleus.

     

Age effect on expression of myosin heavy and light chain isoforms in suspended rat soleus muscle.

This study was designed to test the hypothesis that myosin heavy (MHC) and light chain (MLC) plasticity resulting from hindlimb suspension (HS) is an age-dependent process. By using an electrophoretic technique, the distribution of MHC and MLC isoforms was quantitatively evaluated in the soleus muscles from 3- or 12-wk-old rats after 1-3 wk of HS treatment was maintained. In normal 12- and 15-wk-old rats, the soleus muscles contained a predominance of MHCI ( approximately 94%) with small amounts of MHCIIa, but not MHCIId or MHCIIb. The suspended muscles of adult rats were characterized by the appearance of MHCIIb and MHCIId, the latter reaching approximately 6% after 3 wk of HS treatment. In contrast to changes in MHC, HS did not induce a transition in the MLC pattern in the soleus muscles from adult rats. Compared with adult rats, in juveniles HS had a much more pronounced effect on the shift toward faster MHC and MLC isoform expression. The soleus muscles of 6-wk-old rats after 3 wk of HS were composed of 37.0% MHCI, 19.1% MHCIIa, 23.7% MHCIId, and 20.2% MHCIIb. Changes in MLC isoforms consisted of an increase in MLC1f and MLC2f concomitant with a decrease in MLC2s. These results indicate the existence of a differential effect of HS on MHC and MLC transitions that appears to be age dependent. They also suggest that the suspended soleus muscles from young rats may acquire the intrinsic contractile properties that are intermediate between those in the normal soleus and typical fast-twitch skeletal muscles.     

Expression of myosin heavy chain isoforms in developing rat muscle spindles

Summary The development of muscle spindles, with respect to the expression of myosin heavy chain isoforms was studied in rat hind limbs from 17 days of gestation up to seven days after birth. Serial cross-sections were labelled with antibodies against slow tonic, slow twitch and neonatal isomyosins, myomesin, laminin and neurofilament protein.At 17–18 days of gestation, a small population of primary myotubes expressing slow tonic myosin were identified as the earliest spindle primordia. These myotubes also expressed slow twitch and, to a lesser extent, neonatal myosin. At 19–20 days of gestation a second myotube became apparent; this staining strongly with anti-neonatal myosin. A day later this secondary myotube acquired reactivity to anti-slow tonic and anti-slow twitch myosins. By birth, a third myotube was present; this staining strongly with anti-neonatal myosin but otherwise unreactive with the other antibodies against myosin heavy chains. Three days after birth a fourth myotube, with identical reactivity to the third one, became apparent. Regional variation in the expression of isomyosins, which was present since birth in the two nuclear bag fibers was further enhanced: the nuclear bag₂ staining strongly with anti-slow tonic and antineonatal in the equatorial region and with decreasing intensity towards the poles, whilst with anti-slow twitch the stainability was low in the equatorial and high in the polar region. The nuclear bag₁ fiber showed a homogeneous staining: high with anti-slow tonic, moderate with anti-neonatal, and displayed stainability to antislow twitch myosin in the polar regions only. No regional variation was found along the chain fiber/myotube. At seven days after birth, the pattern of reactivity was similar to that found in the adult spindles, except for the bag₁ fiber which still expressed neonatal myosin.We show that slow tonic myosin is expressed from early development and it is a reliable marker of developing bag fibers. We suggest that muscle spindles are formed from special cell lineages of which the primary generation myotubes expressing slow tonic myosin represent the primordium of muscle spindles.     

Expression of myosin heavy chain isoforms in developing rat muscle spindles

The development of muscle spindles, with respect to the expression of myosin heavy chain isoforms was studied in rat hind limbs from 17 days of gestation up to seven days after birth. Serial cross-sections were labelled with antibodies against slow tonic, slow twitch and neonatal isomyosins, myomesin, laminin and neurofilament protein. At 17-18 days of gestation, a small population of primary myotubes expressing slow tonic myosin were identified as the earliest spindle primordia. These myotubes also expressed slow twitch and, to a lesser extent, neonatal myosin. At 19-20 days of gestation a second myotube became apparent; this staining strongly with anti-neonatal myosin. A day later this secondary myotube acquired reactivity to anti-slow tonic and anti-slow twitch myosins. By birth, a third myotube was present; this staining strongly with anti-neonatal myosin but otherwise unreactive with the other antibodies against myosin heavy chains. Three days after birth a fourth myotube, with identical reactivity to the third one, became apparent. Regional variation in the expression of isomyosins, which was present since birth in the two nuclear bag fibers was further enhanced: the nuclear bag staining strongly with anti-slow tonic and antineonatal in the equatorial region and with decreasing intensity towards the poles, whilst with anti-slow twitch the stainability was low in the equatorial and high in the polar region. The nuclear bag fiber showed a homogeneous staining: high with anti-slow tonic, moderate with anti-neonatal, and displayed stainability to anti-slow twitch myosin in the polar regions only. No regional variation was found along the chain fiber/myotube.(ABSTRACT TRUNCATED AT 250 WORDS)     

NFATc1 and slow-to-fast transition of myosin heavy chain isoforms in gravitational unloading of the rat soleus

An attempt was made to determine whether or not the concentration of NFATc1 (nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1) in nuclear and cytoplasmic extracts is related to an increase in the concentration of fibers containing type IIa myosin heavy chains under modeled gravitational unloading of m. soleus. Experiments were carried out on Wistar rats using the Morey-Holton tail suspension model. It was found that the soleus contains three isoforms of NFATc1 (140, 110, and 86 kDa). Under unloading, the 140-kDa isoform is translocated into the nucleus, the concentration of the 110-kDa isoform in the cytoplasmic extract decreases, and the concentration of the 86-kDa isoform in the nuclear extract increases. Under gravitational unloading of the muscle, the concentration of fibers containing type IIa myosin heavy chains increases. The increase in the concentration of the 140-and 86-kDa NFATc1 isoforms in the nucleus is accompanied by a decrease in the fraction of muscle fibers containing type I myosin heavy chains and an increase in the fraction containing type IIa chains.     

Diversity in expression of myosin heavy chain isoforms and M-band proteins in rat muscle spindles

The composition of adult rat soleus muscle spindles, with respect to myosin heavy chain isoforms and M-band proteins, was studied by light-microscope immunohistochemistry. Serial sections were labelled with antibodies against slow tonic, slow twitch, fast twitch and neonatal myosin isoforms as well as against myomesin, M-protein and the MM form of creatine kinase. Intrafusal fiber types were distinguished according to the pattern of ATPase activity following acid and alkaline preincubations. Nuclear bag1 fibers were always strongly stained throughout with anti-slow tonic myosin, were positive for anti-slow twitch myosin towards and in the C-region but were unstained with anti-fast twitch and anti-neonatal myosins. The staining of nuclear bag2 fibers was in general highly variable. However, they were most often strongly stained by anti-slow tonic myosin in the A-region and gradually lost this reactivity towards the poles, whereas a positive reaction with anti-slow twitch myosins was found along the whole fiber. Regional staining variability with anti-neonatal and anti-fast myosins was apparent, often with decreasing intensity towards the polar regions. Nuclear chain fibers showed strong transient reactivity with anti-slow tonic myosin in the equatorial region, did not react with anti-slow twitch and were always evenly stained by anti-fast twitch and anti-neonatal myosins. All three intrafusal fiber types were stained with anti-myomesin. Nuclear bag1 fibers lacked staining for M-protein, whereas bag2 fibers displayed intermediate staining, with regional variability, often increasing in reactivity towards the polar regions. Chain fibers were always strongly stained by anti-M-protein. The MM form of creatine kinase was present in all three fiber types, but bag1 fibers were less reactive and clear striations were not observed, in contrast to bag2 and chain fibers. Out of 38 cross sectioned spindles two were found to have an atypical fiber composition (lack of chain fibers) and a rather diverse staining pattern for the different antibodies tested. Taken together, the data show that in adult rat soleus, slow tonic and neonatal myosin heavy chain isoforms are only expressed in the muscle spindle fibers and that each intrafusal fiber type has a unique, although variable, composition of myosin heavy chain isoforms and M-band proteins. We propose that both motor and sensory innervation might be the determining factors regulating the variable expression of myosin heavy chain isoforms and M-band proteins in intrafusal fibers of rat muscle spindles.     

Diversity in expression of myosin heavy chain isoforms and M-band proteins in rat muscle spindles

Summary The composition of adult rat soleus muscle spindles, with respect to myosin heavy chain isoforms and M-band proteins, was studied by light-microscope immunohistochemistry. Serial sections were labelled with antibodies against slow tonic, slow twitch, fast twitch and neonatal myosin isoforms as well as against myomesin, M-protein and the MM form of creatine kinase. Intrafusal fiber types were distinguished according to the pattern of ATPase activity following acid and alkaline preincubations.Nuclear bag₁ fibers were always strongly stained throughout with anti-slow tonic myosin, were positive for anti-slow twitch myosin towards and in the C-region but were unstained with anti-fast twitch and anti-neonatal myosins. The staining of nuclear bag₂ fibers was in general highly variable. However, they were most often strongly stained by anti-slow tonic myosin in the A-region and gradually lost this reactivity towards the poles, whereas a positive reaction with anti-slow twitch myosins was found along the whole fiber. Regional staining variability with antineonatal and anti-fast myosins was apparent, often with decreasing intensity towards the polar regions. Nuclear chain fibers showed strong transient reactivity with anti-slow tonic myosin in the equatorial region, did not react with anti-slow twitch and were always evenly stained by anti-fast twitch and anti-neonatal myosins. All three intrafusal fiber types were stained with anti-myomesin. Nuclear bag₁ fibers lacked staining for M-protein, whereas bag₂ fibers displayed intermediate staining, with regional variability, often increasing in reactivity towards the polar regions. Chain fibers were always strongly stained by anti-M-protein. The MM form of creatine kinase was present in all three fiber types, but bag₁ fibers were less reactive and clear striations were not observed, in contrast to bag₂ and chain fibers. Out of 38 cross sectioned spindles two were found to have an atypical fiber composition, (lack of chain fibers) and a rather diverse staining pattern for the different antibodies tested.Taken together, the data show that in adult rat solcus, slow tonic and neonatal myosin heavy, chain isoforms are only expressed in the muscle spindle fibers and that each intrafusal fiber type has a unique, although variable, composition of myosin heavy chain isoforms and M-band proteins. We propose that both motor and sensory innervation might be the determining factors regulating the variable expression of myosin heavy chain isoforms and M-band proteins in intrafusal fibers of rat muscle spindles.     

Expression of myosin heavy chain isoforms along intrafusal fibers of rat soleus muscle spindles after 14 days of hindlimb unloading.

Morphological, contractile, histochemical, and electrophoretical characteristics of slow postural muscles are altered after hindlimb unloading (HU). However, very few data on intrafusal fibers (IFs) are available. Our aim was to determine the effects of 14 days of hindlimb unloading on the morphological and immunohistochemical characteristics of IF in rat soleus muscle. Thirty-three control and 32 unloaded spindles were analyzed. The number and distribution of muscle spindles did not appear to be affected after unloading. There was no significant difference in number, cross-sectional area, and histochemical properties of IF between the two groups. However, after unloading, a significant decrease in slow type 1 MHC isoform and a slight increase in slow-tonic MHC expression were observed in the B and C regions of the bag1 fibers. The alpha-cardiac MHC expression was significantly decreased along the entire length of the bag2 fibers and in the B and C regions of the bag1 fibers. In 12 muscle spindles, the chain fibers expressed the slow type 1 and alpha-cardiac MHC isoforms over a short distance of the A region, although these isoforms are not normally expressed. The most striking finding of the study was the relative resistance of muscle spindles to perturbation induced by HU.     

Skeletal muscle myosin heavy chain isoforms and energy metabolism after clenbuterol treatment in the rat

Prolonged treatment with the beta(2)-adrenoceptor agonist clenbuterol (1-2 mg. kg body mass(-1). day (-1)) is known to induce the hypertrophy of fast-contracting fibers and the conversion of slow- to fast-contracting fibers. We investigated the effects of administering a lower dose of clenbuterol (250 microgram. kg body mass(-1). day (-1)) on skeletal muscle myosin heavy chain (MyHC) protein isoform content and adenine nucleotide (ATP, ADP, and AMP) concentrations. Male Wistar rats were administered clenbuterol (n = 8) or saline (n = 6) subcutaneously for 8 wk, after which the extensor digitorum longus (EDL) and soleus muscles were removed. We demonstrated an increase of type IIa MyHC protein content in the soleus from approximately 0.5% in controls to approximately 18% after clenbuterol treatment (P < 0.05), which was accompanied by an increase in the total adenine nucleotide pool (TAN; approximately 19%, P < 0.05) and energy charge [E-C = (ATP + 0.5 ADP)/(ATP + ADP + AMP); approximately 4%; P < 0.05]. In the EDL, a reduction in the content of the less prevalent type I MyHC protein from approximately 3% in controls to 0% after clenbuterol treatment (P < 0.05) occurred without any alterations in TAN and E-C. These findings demonstrate that the phenotypic changes previously observed in slow muscle after clenbuterol administration at 1-2 mg. kg body mass(-1). day(-1) are also observed at a substantially lower dose and are paralleled by concomitant changes in cellular energy metabolism.